Abstract
Sixteen disulfides derived from disulfiram (Antabuse™) were evaluated as antibacterial agents. Derivatives with hydrocarbon chains of seven and eight carbons in length exhibited antibacterial activity against Gram-positive Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Listeria spp. A comparison of the cytotoxicity and microsomal stability with disulfiram further revealed that the eight carbon chain analog was of lower toxicity to human hepatocytes and has a longer metabolic half-life. In the final analysis, this investigation concluded that the S-octylthio derivative is a more effective growth inhibitor of Gram-positive bacteria than disulfiram and exhibits more favorable cytotoxic and metabolic parameters over disulfiram.
Keywords: disulfiram, disulfides, antibiotic, Staphylococcus, MRSA, VISA, VRSA
Graphical Abstract
Disulfiram (Antabuse™) is an oral prescription drug for the treatment of alcohol abuse disorder [1]. Upon absorption, disulfiram (DSF) [2] and/or its metabolites [3] inhibit aldehyde dehydrogenase (ALDH) enzymes that oxidize acetaldehyde from ethanol metabolism into acetic acid. The inactivation of hepatic ALDH leads to buildup of toxic acetaldehyde in the body, which manifests ‘hangover’ symptoms (e.g., headache, nausea) to deter alcohol consumption [4].
By chemical nature, electrophilic (δ+) DSF is readily cleaved by thiol-bearing substances such as cysteine enzymes. The thiol-disulfide exchange reactions result in the simultaneous addition and release of diethyldithiocarbamate (DDTC). In the case of ALDH, in vitro studies have shown that a second cysteine residue near the addition site may cleave the labile DDTC adduct with concomitant intramolecular disulfide bond formation (Figure 1) [2]. As a versatile inhibitor of cysteine enzymes, DSF has been evaluated as a treatment for other clinical conditions. Recent U.S. clinical trials using repurposed DSF in treatment include: methamphetamine dependence (NCT00731133); cocaine addiction (NCT00395850); melanoma (NCT00256230); muscle atrophy in pancreatic cancer (NCT02671890); and HIV infection (NCT01286259) [5]. In the area of infectious disease, we recently established that DSF inhibits the in vitro growth of methicillin-resistant Staphylococcus aureus (MRSA) at a minimum inhibitory concentration (MIC) range of 4 – 32 μg/mL and exhibits synergism with vancomycin (VAN) against VAN-resistant S. aureus (VRSA) [6]. The mechanism of MRSA inhibition was also attributed to the transfer of DDTC from DSF to thiophilic substances involved in the regulation of bacterial cell growth. Due to its labile chemical nature, we hypothesized that replacement of the DDTC component in DSF (Figure 1) with S-alkylthio groups would increase antibacterial activity and metabolic stability.
Figure 1.
Proposed route of in vitro aldehyde dehydrogenase (ALDH) inactivation by disulfiram [2].
To test this hypothesis, we first synthesized sixteen DSF-derived asymmetric disulfides (1a-p) to deduce the relationship of structure on antibacterial activity (Scheme 1). The compounds were readily prepared by a thiol-disulfide exchange reaction between DSF and respective thiol in DMF [7]. Purification by silica gel chromatography afforded the products as nonaromatic oils in a yield range of 32 – 67% and median yield of 53%. Spectroscopic data and physical characteristics of the compounds were in agreement with previous findings [8].
Scheme 1.
Synthesis of disulfides 1 from disulfiram via a thiol-disulfide exchange reaction.
Antibacterial testing was performed by the broth microdilution assay in 96-well plate format [9,10]. The test agents were initially evaluated against Staphylococcus, Streptococcus, and Enterococcus spp. as our previous research on DSF indicated that Gram-positive cocci would be susceptible [6]. Table 1 shows the MICs of analogs 1a-p in comparison with DSF and VAN. MRSA and Staphylococcus epidermidis exhibited the greatest overall susceptibility to the DSF analogs followed by group A Streptococcus pyogenes (GAS), VAN-resistant Enterococcus faecium (VRE), Streptococcus pneumoniae (SP), and group B Streptococcus agalactiae (GBS). For VISA and VRSA variants of MRSA, the S-heptyl (1i) and S-octyl (1j) derivatives displayed equal or greater antibacterial activity than DSF and VAN.
Table 1.
Susceptibility of Gram-positive cocci to disulfides 1.
| test agent | speciesa | MIC (μM) | |||||||
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| MRSA | VISA | VRSA | VISE | GAS | GBS | SP | VRE | |
| 1a | 32 | 8 | 16 | 32 | 32 | >32 | >32 | >32 |
| 1b | 16 | 8 | 16 | 32 | 32 | >32 | >32 | >32 |
| 1c | 16 | 8 | 16 | 32 | 32 | >32 | >32 | >32 |
| 1d | >32 | 32 | >32 | >32 | 32 | >32 | >32 | >32 |
| 1e | 16 | 16 | 16 | 32 | 32 | >32 | >32 | >32 |
| 1f | >32 | >32 | >32 | >32 | 32 | >32 | >32 | >32 |
| 1g | 16 | 8 | 16 | 8 | >32 | >32 | >32 | >32 |
| 1h | 16 | 4 | 8 | 8 | >32 | >32 | >32 | >32 |
| 1i | 4 | 2 | 4 | 4 | 16 | >32 | 32 | 8 |
| 1j | 4 | 4 | 4 | 4 | 16 | 32 | 16 | 8 |
| 1k | 8 | 8 | 16 | 4 | 8 | 32 | 8 | 8 |
| 1l | 16 | 16 | 32 | 16 | 4 | 16 | 4 | 16 |
| 1m | 32 | 16 | 32 | 32 | 32 | >32 | >32 | >32 |
| 1n | 32 | 16 | 32 | 32 | 16 | >32 | >32 | >32 |
| 1o | 8 | 2 | 8 | 8 | >32 | >32 | >32 | 32 |
| 1p | 32 | 16 | 16 | 16 | >32 | >32 | >32 | >32 |
| disulfiram | 32 | 8 | 32 | 32 | 16 | >32 | >32 | >32 |
| vancomycin | 1 | 4 | >32 | 8 | ≤0.5 | ≤0.5 | ≤0.5 | >32 |
methicillin-resistant Staphylococcus aureus COL (MRSA); vancomycin-intermediate resistant S. aureus ADR-217 (VISA); vancomycin-resistant S. aureus HIP14300 (VRSA); vancomycin-intermediate Staphylococcus epidermidis NRS6 (VISE); group A Streptococcus pyogenes H293 (GAS); group B Streptococcus agalactiae SGBS005 (GBS); Streptococcus pneumoniae TCH8431 (SP); vancomycin-resistant Enterococcus faecium ATCC 700221 (VRE).
The data in Table 1 further reveals a distinct correlation between the length of the S-alkylthio chain and antibacterial activity against Gram-positive cocci. Alkyl chains of seven (1i) and eight (1j) carbons were optimal lengths for antistaphylococcal activity with a MIC of 2 – 4 μM (0.6 – 1.2 μg/mL). By comparison, the MIC ranges of DSF and VAN were 8 – 64 μM (2.4 – 19 μg/mL) and 1 – >32 μM (1.5 – >48 μg/mL), respectively. Short straight chain analogs of one to five carbons were less active than their longer chain counterparts, but were more effective growth inhibitors of MRSA than DSF. Branch and cyclic carbon chain disulfides 1d, 1f, 1m, and 1n similarly had lower activity compared to the straight chain analogs and their respective unbranched equivalents 1c, 1e, 1g, and 1h.
Additional antibacterial testing of the compounds included the select agents Bacillus anthracis (anthrax), Francisella tularensis (tularemia) and Yersinia pestis (plague). Gram-positive B. anthracis exhibited the highest sensitivity to the DSF analogs followed by Gram-negative F. tularensis and Y. pestis (Table 2). In B. anthraces, it was noteworthy that a definitive structure-activity relationship could not be established for analogs with alkyl chains of one to eight carbons in length as seen in S. aureus. Moreover, DSF exhibited greater overall activity for all B. anthraces strains, but not to comparator ciprofloxacin (CIP), which was also the superior test agent against Y. pestis and F. tularensis.
Table 2.
Antibacterial activity of disulfides 1, disulfiram (DSF), and ciprofloxacin (CIP) against select agents.
| species | strain | MIC (μM) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||
| 1a | 1c | 1g | 1h | 1i | 1j | 1o | DSF | CIP | ||
| Bacillus anthracis | Ames35 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 1 | 0.5 |
| Sterne 34F2 | 4 | 4 | 4 | 4 | 2 | 2 | 4 | 2 | ≤0.5 | |
| UM23-1 | 2 | 4 | 4 | 2 | 1 | 1 | 2 | 1 | ≤0.5 | |
| Weybridge | 4 | 4 | 8 | 8 | 2 | 2 | 4 | 2 | 0.5 | |
| Francisella tularensis | Utah 112 | 32 | 32 | 32 | 32 | 16 | 16 | 32 | 32 | ≤0.5 |
| Yersinia pestis | Kim (D2) | 16 | 16 | 32 | 32 | 32 | 32 | 16 | 32 | ≤0.5 |
| Kuma (D7) | 16 | 32 | 32 | 32 | 32 | 32 | 32 | 32 | ≤0.5 | |
To further delineate the antibacterial activity spectrum, the compounds were tested on nineteen additional Gram-positive (n = 5) and Gram-negative (n = 14) species. Table 3 shows that the inhibitory activity was confined to Gram-positive bacteria with Bacillus cereus exhibiting the greatest susceptibility followed by another rod-shaped species, Listeria monocytogenes. Similar to B. anthraces, activity was not predicated on chain length in B. cereus; however, chain lengths of seven (1i) and eight (1j) carbons were the most effective inhibitors of L. monocytogenes as observed with S. aureus. Micrococcus luteus and Rhodococcus erythropolis were also moderately susceptible at a MIC range of 16 – 32 μM. Conversely, the Gram-negative species panel as a whole displayed negligible susceptibility to the analogs. Based on the overall test results, it was concluded that DSF and analogs 1 possess similar narrow-spectrum profiles and straight chain derivatives of seven and eight carbons in length are the most potent growth inhibitors of Gram-positive cocci.
Table 3.
Antibacterial activity of disulfides 1 against Gram-positive and Gram-negative species.
| Gram-positive | -negative | strain | MIC (μM) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||
| 1a | 1c | 1g | 1h | 1i | 1j | 1o | DSF | CIP | ||
| Bacillus cereus | Gibson 971 | 1 | 1 | 1 | 2 | 1 | 1 | 2 | 4 | 0.5 |
| Corynebacterium striatum | FS-1 | >32 | >32 | 32 | 16 | 16 | 16 | 32 | >32 | >32 |
| Listeria monocytogenes | Gibson | 32 | 16 | 8 | 8 | 4 | 4 | 8 | >32 | 4 |
| Micrococcus luteus | SK58 | 32 | 16 | 16 | 16 | 16 | >32 | 16 | 32 | 4 |
| Rhodococcus erythropolis | SK121 | 16 | 16 | 16 | 8 | 16 | 16 | 16 | 16 | ≤0.5 |
|
| ||||||||||
| Acinetobacter baumannii | AB5075 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤0.5 |
| Brucella neotomae | 5K33 | >32 | >32 | 32 | >32 | >32 | >32 | 32 | >32 | 1 |
| Burkholderia cepacia | UCB 717 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 4 |
| Burkholderia multivorans | CF2 | 32 | 32 | >32 | >32 | >32 | >32 | 32 | 32 | 8 |
| Citrobacter freundii | 4_7_47CFAA | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤0.5 |
| Escherichia coli | DC10B | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤0.5 |
| Klebsiella pneumoniae | 700603 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 2 |
| Proteus mirabilis | HM-752 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤0.5 |
| Pseudomonas aeruginosa | 15442 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 1 |
| Salmonella typhi | Ty2 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤0.5 |
| Shigella dysenteriae | Newcastle 1934 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 4 |
| Vibrio cholera | TS (D4) | 32 | 32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤0.5 |
| Yersinia enterocolitica | WA-314 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | 2 |
| Yersinia pseudotuberculosis | P61 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | ≤0.5 |
With a potential application as a treatment for resistant staphylococcal infections, analog 1j was further evaluated for synergistic potential in comparison with DSF. The MICs of different VAN-1j and VAN-DSF concentration combinations were determined using the checkerboard microdilution assay in 96-well plate format [11,12]. Isobologram analysis revealed that analog 1j and DSF lowered the MIC of VAN in MRSA, VISA, and VISE by comparable additive effects (Table 3) [13]. In VRSA, a synergistic effect was observed for both analog 1j and DSF. From these studies it was concluded that disulfide 1j and DSF can similarly lower the MIC of VAN and, therefore, both may have therapeutic utility as antibiotic adjuvants for staphylococcal infections with reduced VAN susceptibility.
Table 3.
Results of in vitro synergy studies.
| strain | MIC (μg/mL)a | ΣFICd | |||||
|---|---|---|---|---|---|---|---|
|
|
|
||||||
| VAN | DSF | 1j | VANb/DSFc | VANb/1j | VANb/DSFc | VANb/1jc | |
| MRSA COL | 2 | 8 | 2 | 1/1 | 1/0.25 | 0.63 (+) | 0.75 (+) |
| VISA AR-217 | 4 | 4 | 0.5 | 2/1 | 0.5/0.25 | 0.75 (+) | 0.56 (+) |
| VRSA HIP14300 | >128 | 16 | 2 | 8/2 | 1/1 | <0.16 (++) | 0.5 (++) |
| VISE NRS53 | 8 | 4 | 2 | 4/2 | 2/1 | 1 (+) | 0.75 (+) |
vancomycin: VAN; disulfiram: DSF
lowest MIC of VAN in combination with DSF or 1j
lowest MIC of DSF or 1j in combination with VAN
lowest ΣFIC measurement; synergy (++) ≤ 0.5; additive (+) 0.5 < to ≤ 1; indifferent (±) 1< to ≤ 4; antagonism (−) > 4 [12]
The investigation also compared the cytotoxicity of the analogs with DSF in human liver HepG2 carcinoma cells using the MTT assay [14]. The dose-response curve in Figure 2 revealed that the half-maximal inhibitory concentrations (IC50) of analogs 1c (51 μM), 1h (51 μM), 1j (50 μM), and 1k (55 μM) were above DSF (38 μM), thereby indicating lower cytotoxicity. The >13:1 ratio of S. aureus MIC (4 μM) to IC50 further suggests that compound 1j is selectively toxic to the bacterium. The apparent selectivity was partially attributed to the higher glutathione content in mammalian cells [15]. The thiophilic tripeptide, which is found in low abundance in Gram-positive bacteria, has been shown to inactivate disulfide-based antibacterials through a thiol-disulfide exchange reaction [6,14,16].
Figure 2.
Dose-response curve for liver HepG2 cells.
Additional studies of analog 1j included comparisons of microsomal stability and physiochemical properties to DSF (Table 4). Measurement of the in vitro metabolic stability using pooled rat liver microsomes indicated that the elimination half-life (t½) and intrinsic clearance (CLint) was longer for the DSF analog [17,18]. A comparison of the physiochemical chemical properties also revealed that both DSF and derivative 1j are hydrophobic compounds. The higher clogP and lower molecular polar surface area (PSA) for disulfide 1j suggest that replacement of the DDTC component in DSF with the more lipophilic S-octylthio group may confer better tissue and membranes penetration [19]. This marked difference with DSF could partially account for the increased susceptibility of S. aureus to analog 1j if cell entry is required of both agents to inhibit growth.
Table 4.
Microsomal stability and calculated physiochemical properties.
In the final analysis, S-alkylthio analogs of DSF exhibited up to eight times greater antibacterial activity compared to DSF. The activity spectrum of the analogs was similar to DSF with Gram-positive Staphylococcus and Bacillus spp. exhibiting the highest level of susceptibility. Analogs with S-heptylthio (1i) and S-octylthio (1j) groups were found to be the most effective inhibitors of MRSA growth and retained their potency against VISA and VRSA. The select antibacterial activity was partly attributed to the low abundance of redox-buffering glutathione in the cytoplasm of Gram-positive bacteria [14]. Glutathione has been shown to inactivate disulfide-based antibacterial agents [6,14,16] and the higher abundance in Gram-negative bacteria may account for the Gram-type selectivity. This investigation also considered that the outer membrane barrier in Gram-negative bacteria could be a factor; however, the data from Table 3 indicated that increasing the lipophilic property of the compounds, which would facilitate cell membrane permeation, did not effect antibacterial activity in Gram-negative bacteria. Other factors that would account for the Gram-type selectivity, but were not investigated during the study, are the existence of potential pharmacological targets and cellular pathways required for antibacterial action.
This research further resolved that disulfide 1j can lower the MIC of VAN in VISA and VISE, suggesting possible therapeutic utility as an antibiotic adjuvant in VAN-intermediate infections. Preliminary assessment of cytotoxicity and microsomal stability in comparison with DSF also confirmed that the S-octylthio analog was of lower toxicity to human hepatocytes and had a longer half-life, a parameter with implications on dosing frequency. Future research will focus on defining the mechanisms of action and resistance development for disulfide-based antibacterials. In addition, pharmacokinetic-pharmacodynamic (PK-PD) studies will be performed in vivo to determine their viability as antibiotic adjuvants in VAN therapy. The PK studies will be used to guide dosing with a regimen that accounts for the influence of tissue glutathione on the disulfide concentration and gives a preferred trough VAN concentration of 4 to 5 times the MIC [22] with the disulfide for the test pathogen (e.g., VISA).
Acknowledgments
This research was supported by the Marshall University School of Pharmacy FRS Grant Program. The authors also thank the Marshall University Department of Chemistry for use of the NMR facility. Bacteria were acquired from the American Type Culture Collection, FDA-CDC Antimicrobial Resistance Isolate Bank, and the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH.
Footnotes
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References
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